Function And Mechanism Study Of RNF113A In Esophageal Squamous Cell Carcinoma

Objective: To investigate the role of RNF113 A in the malignant behavior of Esophageal squamous cell carcinoma(ESCC)including the ability of apoptosis,proliferation,migration,and invasion and its possible mechanism;and to analyze the clincopathological significance in Kazakh’s ESCC tissues.To explore whether RNF113 A could become one of the molecular targets for diagnosis and treatment of Kazakh’s esophageal cancer.Methods: 1)To investigate the clinicopathological significance of RNF113 A expression in RNF113 A ESCC and paired normal control tissues,immunohistochemistry was employed contained 117 cases of Kazakh’s ESCC tissues,and the relationship between the expression of RNF113 A and p53 was analyzed.Real-time quantitative PCR(qRT-PCR)was performed to detect the expression in 41 Kazakh’s ESCC tissues and paired normal control tissues.Analyze the relationship of the expression of RNF113 A and the clinicopathological parameters include patients “age,gender,differentiation,T classification,N classification,tumor location,lymph node metastasis,gross pathology”.The relationship between RNF113 A expression and prognosis of patients was analyzed.2)To investigate the role of RNF113 A in the proliferation,migration,invasion,cell cycle and apoptosis of ESCC cell line Eca109 cells.First of all,specific siRNA interference vectors against RNF113 A and eukaryotic over-expression vectors harboring full-length c DNA of RNF113 A were constructed.Followed by transfection into ESCC cell line.q RT-PCR and western-blot were used to detect the mRNA and protein levels of RNF113 A after transfection.To detect the cell cycle and apoptotic variation before and after transfection with siRNA and eukaryotic over-expression vectors into ESCC cells,Flow Cytometry(FCM)was utilized.To evaluate the influence over migratory and invasive variation,wound-healing and Transwell assays were used.3)To analyze the tumorigenic ability of RNF113 A,transgenic cell line Eca109 whose basal RNF113 A was stably knocked down were established.Normal Eca109 cell,Eca109 cells transfected with Lentiviral-shRNA-RNF113 A or sh-scramble lentivirus were injected to nude mice.Tumor volumes and body weights were measured every week.Tumor volumes were calculated with the the equation: volume=(longest diameter×shortest diameter2)/2.We used qRT-PCR and immunohistochemistry assay to detect the expression of RNF113 A in mice tumor mass organization.Results: Part one: RNF113 A expression was positively detected in 63.25%(74 of 117)in Kazakh’s ESCC samples and RNF113 A was up-regulated in ESCC tissues in comparison with paired normal control tissues(P=0.000).Statistical analysis showed a significantly positive correlation between RNF113 A and p53 expressions in the ESCC tissues tested(P=0.035).RNF113 A mRNA expression in 41 fresh Kazakh’s ESCC was two times increased compared with the adjacent non-tumor tissues.RNF113 A expression was correlated with differentiation degree(P=0.008),T classification(P=0.000),lymph node metastasis(P=0.017).There was no significant difference between RNF113 A expression and clinicopathological parameters,including gender,age,gross classification and tumor location.Kaplan-Meier survival analysis indicated the high expression of RNF113 A was significantly associated with poorer overall survival.In univariate analysis showed RNF113 A expression,T classification and lymph node metastasis were significantly associated with the overall survival time.The final multivariate model revealed that overall survival time significantly depended on RNF113 A expression and lymph node metastasis.Part two: Cell number was significantly increased in the G0/G1 phase and decreased in the S phase and G2/M phase after being knocked down in ESCC cell lines;Cell number was significantly decreased in the G0/G1 phase and increased in the G2/M phase after being overexpressed in ESCC cell lines.Apoptosis assay showed that the apoptosis was increased when RNF113 A were knocked down;Overexpression of RNF113 A could also lead to apoptosis to a certain extent,but there was no significant difference compared to empty vector group.RNF113 A was able to inhibate the migration and invasion ability in ESCC cells after being knocked down;Overexpression of RNF113 A can promote the migration and invasion of ESCC cells.Part three: Results from nude mice xenografted with Eca109 showed that the tumor volume and weight were smaller and lighter after knock-down of RNF113 A.It suggests RNF113 A was capable of increasing the progression of Eca109 cells in vivo.Conclusion: RNF113 A expression is high expression in Kazakh’s esophageal cancer,and was correlated with differentiation degree,T classification,lymph node metastasis and overall survival.RNF113 A inhibits the cell apoptosis,promotes proliferation,migration and invasion,and influence cell cycle distribution of esophageal squamous cell carcinoma.In xenografted nude mice,RNF113 A was able to promote the progression in vivo,supports its oncogenic role in ESCC.